The long term goal of this project is to identify and characterize the molecular mechanisms of insulin action on monosaccharide transport in cardiac tissue. The goal of this proposal is to investigate several current hypotheses relating to this effect of insulin: 1) The recently proposed hypothesis that insulin induces a translocation of glucose transport systems from a cellular cytosolic pool into the plasma membrane, and 2) the possible role of nucleotides in regulating insulin effects on sugar transport at the level of the plasma membrane. We will use a method recently developed in our laboratory for preparing insulin-sensitive, isolated cardiac myocytes as a model to study several possible mechanisms for control of this hormonally induced process. In addition, we will utilize another methodology which we have recently developed to study the molecular components associated with the glucose transport system. This method irreversibly labels the glucose transporter in erythrocytes by photoincorporation of (3H)cytochalasin B, a potent inhibitor of this transport system. We will attempt to extend this method to label and identify the transport-associated protein(s) of the cardiac myocyte, in both the plasma membrane as well as any additional cellular subfractions. We will also utilize immunological methods to isolate, purify and reconstitute this transport system from these cells. We hope this method will be useful in testing the "transporter-translocation" hypothesis, as well as in identifying the actual proteins involved in that process. These studies may additionally provide a basis for analyzing glucose transport and the effects of insulin on this system in both the normal and disease states of cardiac tissue.